Biochemical and structural studies of metallo-β-lactamase superfamily enzymes

The metallo-β-lactamase (MBL) superfamily is characterised by a unique αββα structural fold and is widespread in nature. Whilst best known for its name-lending members that hydrolyse β-lactam antibiotics and contribute to antimicrobial resistance, other members of the MBL superfamily also utilise metal ions to catalyse various hydrolysis and redox reactions. The work described in this thesis focuses on characterisation of two MBL proteins – cytidine monophosphate N acetylneuraminic acid hydroxylase (CMAH) and glyoxalase II (GloB). CMAH catalyses hydroxylation of a sialic acid cytidine monophosphate N-acetylneuraminic acid (CMP Neu5Ac) to CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) in the presence of O2, cytochrome (cyt) b5, cyt b5 reductase and NADH and is found in animals of the Deuterostomia lineage. Human (h) CMAH is catalytically inactive due to a deletion which occurred approximately 3 million years ago. Understanding the differences between hCMAH and its catalytically active homologues could provide insight into the CMAH mechanism and the function of hCMAH in humans. In this thesis, development of expression systems for hCMAH and its active mouse homologue mCMAH, their initial characterisation and efforts to obtain the first CMAH structure are described. Despite the lack of catalytic activity of hCMAH, the human CMAHP gene is transcribed in cells suggesting an alternative function. Studies on the cellular localisation of hCMAH revealed cytoplasmic localisation with some compartmentalisation being observed. Additionally, attempts were made to detect proteins interacting with hCMAH and mCMAH in mammalian cells. GloB is involved in the methylglyoxal detoxification pathway. GloB catalyses hydrolysis of S-D-lactoylglutathione to glutathione and D-lactate. The thesis described work on the characterisation of GloB from Escherichia coli. Kinetic studies using various transition metals are described as well as the search for potential inhibitors. Crystal structures of GloB at 1.1 Å and 1.8 Å resolution are reported and initial attempts to obtain the structure of an enzyme substrate complex are described. Overall, the work described in this thesis explores the biochemical, functional and structural features of two very different MBL superfamily proteins and provides insights into the scope of differences present within the superfamily.